The present invention relates to the therapy of bone disorders associated with reduced bone mass.
Bone formation, the synthesis and deposition of extracellular matrix, is essential for skeletal growth, modeling, remodeling and repair. Osteoblasts, the bone-forming cell type of the skeleton, originate from pluripotent mesenchymal stem cells. The differentiation and proliferation of osteoblasts can be modulated by numerous extracellular factors such as hormones, growth factors and cytokines. Recently, the transcription factor Cbfa-1 was found to be essential for osteoblast differentiation (Banerjee, et al., 1997; Ducy, et al., 1997; Otto, et al., 1997; Komori, et al., 1997). However, the molecular mechanisms which control bone formation in vivo are poorly understood.
Activator protein-1 (AP-1) is a dimeric transcription factor composed of Jun, Fos or ATF (activating transcription factor) family members. AP-1 binds to a common DNA site, the AP-1 binding site, and converts extracellular signals into changes in the transcription of many cellular and viral genes (reviewed in Angel and Karin, 1991). AP-1 activity is modulated by various signals including growth factors, cytokines, tumor promoters, carcinogens and specific oncogenes. AP-1 has been implicated in a number of biological processes such as cell proliferation, cell differentiation and apoptosis. However, analysis of AP-1 functions in vivo and in tissue culture cells have shown that different AP-1 members regulate different target genes and thus execute distinct biological functions in a cell-type specific fashion.
Several lines of evidence suggest that AP-1 participates in the control of osteoblast functions. Consensus AP-1 DNA binding sites are present in the promoter regions of genes involved in the regulation of osteoblast growth, differentiation, and extracellular matrix formation and degradation, such as alkaline phosphatase, type I collagen, osteocalcin, osteopontin, and matrix metalloproteases-1 and -13 (Owen, et al., 1990; Schule, et al., 1990; Guo, et al., 1995; Angel, et al., 1987; Pendas, et al., 1997). A number of regulators of osteoblast proliferation and differentiation, including transforming growth factor-xcex2 (TGF-xcex2), parathyroid hormone, growth hormone and 1,25-dihydroxyvitamin D, induce the expression of AP-1 components in vitro and in vivo in osteoblastic cells (Candeliere, et al., 1991; Slootweg, et al., 1991; Clohisy, et al., 1992; Machwate, et al., 1995; Koe, et al., 1997). Moreover, the various components of the AP-1 complex are differentially expressed during osteoblast differentiation in vitro and can be detected at sites of active bone formation in vivo (Dony and Gruss, 1987; Sandberg, et al., 1988; Smeyne, et al., 1992; McCabe, et al., 1995; McCabe, et al., 1996).
Fra-1 is an immediate early gene encoding one member of the AP-1 family of transcription factors which shows extensive amino acid homology to c-Fos (Cohen and Curran, 1988). Fra-1 forms heterodimeric complexes with all Jun proteins (c-Jun, junB, junD) and interacts with AP-1 binding sites to regulate gene transcription (Cohen, et al., 1989; Ryseck and Bravo, 1991; Suzuki, et al., 1991). Unlike c-Fos, Fra-1 lacks a C-terminal transactivation domain (Wisdom and Verma, 1993). In addition to induction by serum and mitogens, Fra-1 expression is regulated upon lymphocyte activation and during the differentiation of keratinocytes, spermatocytes and osteoblasts (McCabe, et al., 1995; McCabe, et al., 1996; Cohen and Curran, 1988; Cohen, et al., 1993; Welter and Eckert, 1995; Gandarillas and Watt, 1995; Huo and Rothstein, 1996; Rutberg, et al., 1996). Moreover, ectopic expression of Fra-1 in osteoclast progenitor cell lines potentiates osteoclast development (Owens, et al., 1999).
Reduced bone mass, either circumscribed or systemic, results in impaired bone strength and predisposes to pathological fractures.
Common causes of localized osteolytic lesions are metastatic bone disease, multiple myeloma and lymphoma. In addition, circumscribed bone defects can be caused by numerous benign bone disorders including, among others, bone cysts, fibrous dyslasia, infections, benign bone tumors and impaired fracture healing. Current treatment of these lesions comprises surgical removal or radiotherapeutic destruction of the pathological tissue, fracture fixation, implant stabilization and the reconstruction of the skeletal defect. However, current surgical methods utilizing autograft or allograft bone to close the skeletal defects have limitations. Autograft procedures can result in donor site fracture and donor-site pain, and are limited by the amount of autogenous bone available. Allograft is biologically inactive in the host and has immunological and infectious disease risks.
In contrast, osteoporosis is a systemic disease characterized by low bone mass and microarchitectural deterioration in the entire skeleton with a consequent increase in bone fragility and susceptibility to fracture, especially of bones subjected to major mechanical forces.
Bone is remodeled throughout life, involving the coordinate occurence of bone resorption and bone formation. Osteoporosis develops if the rate of bone resorption exceeds the rate of bone formation resulting in a progressive loss of bone mass. A large number of risk factors for osteoporosis have been identified including aging and loss of gonadal function. In addition, osteoporosis is associated with various endocrine, haematologic, gastrointestinal and rheumatologic diseases, and can be the consequence of therapy with glucocorticoids, heparin, and antiepileptic drugs. The major clinical manifestation of osteoporosis are vertebral body fractures, leading to pain in the back and deformity of the spine. The diagnosis of osteoporosis is based on reduced bone mass, usually assessed by measuring bone mineral density. Most of the drugs used to treat osteoporosis act by decreasing bone resorption, including estrogens, bisphosphonates, and calcitonin. Therapeutic regimens which effectively stimulate bone formation are not available. Although sodium fluoride therapy results in large increases in bone mineral density, its effect on fracture rates is small, since it stimulates the formation of a bone matrix with low mechanical strength.
It was the object of the present invention to provide an improved therapy to restore the mechanical properties of affected bone(s) by enhancing bone formation, either locally or in the entire skeleton, in individuals suffering from bone disorders that are associated with a circumscribed or systemic reduction of bone tissue.
In order to provide a therapeutic approach based on the administration of drugs that are capable of stimulating bone formation, the cellular and molecular mechanisms underlying bone formation were studied.
In order to study the consequences of ectopic Fra-1 expression in vivo, transgenic mice were generated which express high levels of Fra-1 in a broad range of tissues, including bone. It was shown that ectopic Fra-1 expression stimulates bone formation by osteoblasts leading to the development of increased bone mass in the entire skeleton. Furthermore, the data obtained in the experiments of the present invention indicate that constitutive Fra-1 expression promotes osteoblast proliferation and differentiation, since transgenic bones contain increased numbers of mature osteoblasts. Moreover, osteoblastic cells derived from transgenic mice were shown to undergo an accelerated course of differentiation in vitro indicating that Fra-1 can positively regulate bone formation in vivo and in vitro.
Maintenance of bone mass depends on the balance between bone formation by osteoblasts and bone resorption by osteoclasts. In the majority of mouse models of increased bone mass reported on to date, impairment of bone resorption is causal, due to defects either in osteoclast differentiation or function, resulting in osteopetrosis (Johnson, et al., 1992; Wang, et al., 1992; Grigoriadis, et al., 1994; Iotsova, et al., 1997; Simonet, et al., 1997; Tondravi, et al., 1997; Saftig, et al., 1998; Kong, et al., 1999). In contrast, the mouse model of the present invention, i.e. fra-1 transgenic mice, were shown to develop increased bone mass as a consequence of increased bone formation by promoted osteoblast differentiation, rather than decreased bone resorption by osteoclasts. Several lines of evidence support this conclusion. Histomorphometric parameters measuring bone formation were increased in the fra-1 transgenic mice. Grafting experiments revealed that the cause of the bone phenotype resides in transgenic osteoblasts and is therefore not due to secondary effects, e.g. increased hormone secretion: chimeric bones formed by transgenic osteoblasts and wild-type osteoclasts develop increased bone formation, whereas bones composed of wild-type osteoblasts and transgenic osteoclasts display no histological features of increased bone mass. Indirect evidence is provided by the observations that osteoclasts are present in transgenic mice and cover bone surfaces to an comparable extent in transgenic and wild-type mice. Finally, transgenic osteoclasts resorb bone matrix in vitro indicating that reduced resorption function is not the cause for increased bone mass in transgenic skeletons.
To date, the regulation of bone formation by transcription factors has been poorly understood at the molecular level. Beyond its essential function during embryonic development, Cbfa-1 also controls the deposition of bone extracelluar matrix in adult mice (Ducy, et al., 1999). In contrast to Cbfa-1, which regulates the synthesis of extracellular matrix at the level of the single osteoblast, Fra-1 appears to increase the number of active osteoblasts as indicated by increased histomorphometric values for osteoblast surface and mineralizing surface. In contrast, mineral apposition rate and osteoid thickness were comparable in transgenic and wild-type femora, suggesting that the bone forming activity of transgenic osteoblasts is largely unchanged.
Under differentiating conditions in vitro, osteoblastic cells undergo a defined pattern of differentiation characterized by the temporal expression of osteoblast-specific genes. It could be shown by the present inventors that cultures of the transgenic osteoblasts display an accelerated time course of ALP activity and mineralization indicating that Fra-1 expression promotes the differentiation of progenitor cells into bone-forming osteoblasts. The molecular mechanism by which Fra-1 enhances osteoblast differentiation is unknown. Without wishing to be bound by theory, Fra-1 could induce the expression of growth factor receptors or growth factors in osteoblasts. Candidate factors are TGF-xcex21 and the insulin-like growth factor-2, both positive regulators of bone formation in vitro and in vivo, which are considered to be potential AP-1 target genes, since the expression of both genes is inducible by AP-1 (Noda and Camilliere, 1989; Joyce, et al., 1990; Kim, et al., 1990; Caricasole and Ward, 1993). Mice carrying a targeted disruption of the osteocalcin gene show a similar, although milder, bone phenotype raising the possibility that Fra-1 may act by decreasing osteocalcin expression in transgenic osteoblasts (Ducy, et al., 1996). However, osteocalcin expression was slightly increased in the calvariae of transgenic and wild-type mice suggesting that Fra-1 acts independently of osteocalcin. Alternatively, Fra-1 could directly induce positive regulators of the cell cycle leading to increased proliferation within the osteoblast lineage. Interestingly, Fra-1 together with c-Jun are able to upregulate cyclin D1 expression in fibroblasts (Mechta, et al., 1997).
It was shown that osteoblasts display a high susceptibilty to elevated levels of Fra-1 expression. The stimulatory effect on bone formation appears to be specific for Fra-1 in comparison to other members of the AP-1 family of transcription factors. Mice expressing high levels of FosB, Fra-2, c-Jun or JunB in bone tissue show no skeletal phenotype (Grigoriadis, et al., 1993; McHenry, et al., 1998). However, c-Fos, another member of the Fos subfamily of AP-1 factors, displays a similar tropism for the osteoblast lineage. Overexpression of c-Fos in transgenic mice results in osteoblast transformation and the development of skeletal osteosarcomas (Grigoriadis, et al., 1993; Ruther, et al., 1989)
Thus, the finding of the present invention that Fra-1 is a positive regulator of bone formation, provides the basis for using Fra-1 as a target for pharmaceutical intervention in pathological conditions in which bone mass is reduced or bone formation is impaired, e.g. in osteoporosis or impaired fracture healing.
The findings of the present invention can be harnessed for designing assays for identifying compounds that upregulate Fra-1 expression and thus are osteoinductive drug candidates for the treatment of bone disorders.
The present invention is directed to a method for identifying a substance for the treatment of bone disorders associated with reduced bone mass, characterized in that the substance is tested for its ability to modulate the expression of Fra-1 or a Fra-1 target gene in osteoblasts, said upregulation or modulation resulting in an increase of bone mass in vivo.
In a first aspect, the substances are tested for their ability to upregulate the expression of Fra-1.
In an embodiment of the invention, a screening method is provided which is based on mammalian cells, preferably human cells, in which the induction of Fra-1 expression upon incubation with the test substance can be monitored.
This screening method of the invention is based on a type of assay known in the art for identifying compounds that transcriptionally modulate the expression of a gene of interest. It may designed and carried out according to known methods, e.g. as described in WO 91/01379. It is well suited for use in a high-throughput format in order to test a great number of chemical compounds.
In a preferred aspect, the induction of Fra-1 expression upon incubation with the test compound is monitored by determining the expression of a reporter gene under the control of regulatory sequences of the fra-1 gene. The regulatory regions of the rat and murine fra-1 genes are known and easily available for the person skilled in the art (Bergers et al., 1995; Schreiber et al., 1997). They include intragenic sequences in the 5xe2x80x2 region and sequences in the first exon and first intron of the fra-1 gene. An example for a suitable regulatory region for a reporter gene construct is a 5.5 kb fragment of the rat fra-1 gene harboring the 5 flanking sequences, exon 1 and the 5 half of intron1. Alternatively, a 1.6 kb SacI fragment containing the first 710 bp of the 5 flanking sequence, exon 1, and part of intron 1 of the fra-1 gene may be used (Bergers et al., 1995).
Preferably, the regulatory regions of the human fra-1 gene are employed. Based on the murine and rat sequences of the fra-1 gene, the human fra-1 genomic DNA can be cloned by conventional techniques, e.g. by screening human genomic libraries with rat or murine fra-1 probes, and the regulatory sequences can be obtained from the cloned gene.
Alternatively to using the fra-1 regulatory sequences, the regulatory sequence of a fra-1 target gene may be used. Fra-1 target genes, i.e. genes the expression of which is modulated by Fra-1 expression, can be identified, as described below. Target genes, the regulatory regions of which are useful to be employed in a screening assay, are those which, like fra-1, display a stimulatory effect on bone formation. In the case that the Fra-1 effect on bone formation is due to the down-regulation of the Fra-1 target gene of interest, the above-described assay can be used to identify substances which down-regulate the respective Fra-1 target gene by determining the reduced expression of the reporter gene under the control of regulatory sequences of that gene.
In a preferred embodiment, the method of the invention is carried out as follows:
In a first step, suitable test cells are selected, i.e. cells which can be easily grown in tissue culture and in which the activation of a reporter gene can be measured. In a preferred embodiment, primary osteoblasts are used as test cells. Primary osteoblasts can be obtained by methods available in the art, e.g. by the method described in Example 4. Alternatively, osteosarcoma cell lines and osteoblast-like cell lines, e.g. MC 3T3-E1 cells may be employed. Further examples for cell lines are Saos-2 (human; ATCC HTB-85), U-20S (human, ATTC HTB-96), UMR-106 or 108 (rat; ATTC CRL-1661 or ATTC CRL-1663). In principle, any other cell type may be used in which expression of Fra-1 can be induced, e.g. fibroblasts, or PC12 (rat pheochromoytoma) cells, however, the inductive effect of compounds identified in such cells has to be confirmed in osteoblasts. Thus, a screen using cells closely related to osteoblasts is preferred.
The test cells are stably transfected by conventional techniques (Sambrook, et al., 1989), with a recombinant DNA molecule containing the regulatory regions of the fra-1 gene, preferably in combination with a minimal promotor, e.g. the SV40, xcex2-globin or TK minimal promotor sequence, fused to a reporter gene according to known methods. Any reporter gene may be used whose expression product is detectable by measuring a signal proportional to its concentration and which can be detected and quantified. Preferably, a reporter gene is employed with high sensitivity in an automated assay. Examples for preferred reporter genes are the luciferase gene (De Wet, et al., 1987), the Green Fluorescent Protein GFP (Kallunki, et al., 1994), and the lacZ gene. The cells are grown in a suitable medium in microtiter assay plates and incubated with the substances to be tested. Substances which exhibit the desired effect, i.e. the induction of Fra-1 expression, are expected to upregulate reporter activity as compared to identical control cells that are incubated under the same conditions in the absence of the test substance.
For specificity control, control cells are incubated with the test substance under the same conditions as the test cells; the control cells lacking in their reporter gene construct the fra-1 regulatory elements but being otherwise identical to the test cells; substances that will be selected as drug candidates are those which cause an increase in reporter gene expression only in cells with the fra-1 construct. (In the case of using a minimal promotor sequence in the test cells, the reporter gene in the control cells is driven by the minimal promotor sequence only.)
An alternate specificity control uses cells which are identical to or different from the test cells in cell type and contain the reporter gene under the control of a transcriptional control unit different from the fra-1 regulatory element; a test substance that has shown to increase reporter gene expression in the fra-1 test cells should induce no increase in reporter gene expression in such a control cell.
In the case of applying the assay to a Fra-1 target gene which needs to be downregulated by the compound to achieve the desired therapeutic effect, this effect is reflected by reduced expression of the reporter gene upon incubation with the substance.
To optimize the assay, the test cells are incubated, in series experiments, under varying assay conditions, with a substance known to induce Fra-1 expression in cultured cells, e.g the phenolic antioxidant tert-butylhydroquinone (Yoshioka et al., 1995) and the reporter gene expression is measured.
To further evaluate substances identified in the primary screen, secondary screens may be conducted which are based on determining biological effects associated with Fra-1 overexpression. Examples of known biological effects are achorage-independent growth in vitro, invasive growth in vitro and cell motility. Assays to measure these effects are described by Bergers et al., 1995, and Kustikova et al., 1998.
In a next step, the compounds may be tested for their osteoinductive activity, i.e. their ability to accelerate osteoblast differentiation and proliferation in vitro and/or to increase bone formation in vivo by using animal models, e.g.mice.
Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the method of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50, the ED50. The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal). A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. xe2x80x9cRemington Pharmaceutical Sciencesxe2x80x9d. Examples for ingredients that are useful for formulating the compounds identified according to the present invention are also found in WO 99/18193.
The osteoinductive low-molecular compounds or fra-1 DNA molecules can be used in the therapy of circumscribed or systemic bone disorders associated with reduced bone mass, as described above.
For osteoporosis, the therapeutic strategy comprises a treatment with the osteoinductive drug until normal bone mass compared to appropiate control groups is restored. Bone mass can be assessed by determining bone mineral density. Then the treatment can be switched to established regimens for the prevention of bone loss to avoid potential side effects of overshooting bone formation.
For circumscribed bone disorders, a promising therapeutic approach may be placing bone grafts at the site of the lesion and administering fra-1 DNA locally in order to enhance bone formation.
Toxicity and therapeutic efficacy of the substances identified as drug candidates by the method of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50, the ED50. The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal). A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. xe2x80x9cRemington Pharmaceutical Sciencesxe2x80x9d. Examples for ingredients that are useful for formulating the compounds identified according to the present invention are also found in WO 99/18193.
In a further embodiment, the invention relates to fra-1 DNA for therapy, in particular for the treatment of bone disorders. In this embodiment, the DNA is employed by methods known for somatic gene therapy.
xe2x80x9cFra-1 DNAxe2x80x9d designates any DNA molecule encoding Fra-1 or a fragment thereof that has the ability of inducing increased bone formation upon expression in osteoblasts. The DNA may be the entire fra-1 cDNA or genomic fra-1 DNA, a fragment or a mutant thereof, preferably of human origin. To determine the effectiveness of the DNA molecule to be employed in the gene therapy approach, the candidates, e.g. the entire fra-1 cDNA and various fragments or mutants thereof, are transferred into the target cells, i.e. the osteoblasts, by viral or non-viral gene transfer methods known in the art. Examples for viral gene transfer vectors are retroviral or adenoviral vectors, into which the sequence to be tested is inserted under the control of a constituive or inducible promotor. The thus genetically modified cells are cultured under differentiating conditions (see Example 4), and the desired biological activity is determined by monitoring the time course of differentiation and the amount of mineralized extracellular matrix formed in vitro.
For the therapy of humans, the DNA encoding the biologically active Fra-1, is delivered by viral or non-viral gene therapy application routes such that constitutive or inducible expression of Fra-1 in osteoblasts is achieved at a level to achieve sufficient Fra-1 expression and thus the desired therapeutic effect. An example for the gene therapy is the use of retroviral or adenoviral vectors, into which the fra-1 DNA molecule, under the control of a strong promotor, is inserted. To target Fra-1 expression to osteoblasts, the promoter of the osteocalcin gene can be used (Hou et al., 1999). The fra-1 DNA may be administered systemically, e.g. in the case of osteoporosis therapy, or locally, in the case of circumscribed bone defects by applying it to the site of the defect.
In a further embodiment of the invention, transgenic animals, preferably mice, are provided which constitutively express Fra-1 in osteoblasts (fra-1 transgenic animals).
These mice are obtainable by standard technology for the generation of transgenic animals, e.g. by microinjection of the fra-1 transgene containing the murine genomic fra-1 sequence, into pronuclei of one-cell embryos (Hogan, 1994).
The fra-1 transgenic mice of the invention can be used to identify target genes regulated by Fra-1 during bone formation or other biological processes. Primary cells, e.g. osteoblasts, from fra-1 transgenic and wild-type control mice are isolated and differentiated in vitro. Differences in RNA of protein levels are determined by standard methods known in the art for differential gene transcription and/or protein expression, e.g. differential display RT-PCR analysis (Liang and Pardee, 1992; Bauer, et al.,1993), DNA microarray technology (DeRisi, et al., 1997; Schena, et al., 1995; Wodicka, et al., 1997; Ramsay, 1998), subtractive hybridization (Diatchenko, et al., 1996; Hubank and Schatz, 1994), or proteome analysis (Pennington, et al., 1997; Pasquali, et al., 1997).
The present invention provides the first genetic evidence that Fra-1 is a positive regulator of bone formation in vivo. Since Fra-1 overexpression does not appear to interfere with vital physiological functions, drugs that induce Fra-1 expression or Fra-1 itself may be used in the treatment of diseases characterized by reduced bone mass or impaired bone formation, e.g. osteoporosis and impaired bone fracture healing.